Microalgae are small-sized organisms found in fresh and saline waters, in both benthic and littoral habitats, and also throughout the ocean waters as phytoplankton, while the larger macroalgae (seaweeds) occupy the littoral zone (Hasan et al., 2009; El Gamal, 2012). Microalgae are unicellular to filamentous in form. They lack roots, vascular systems, leaves and stems, and are autotrophic and photosynthetic. Microalgae are generally eukaryotic organisms, although cyanobacteria, such as spirulina, which are prokaryotes, are included under microalgae due to their photosynthetic and reproductive properties (Ravishankar et al., 2012). Microalgae range in size from about 5 µm (Chlorella) to more than 100 µm (spirulina) (Becker, 2013b). The commercial cultivation of microalgae began in Japan with the cultivation of Chlorella in the 1960s, followed by the cultivation of spirulina in Mexico and the USA in the 1970s. Since then, the industrial biotechnology of microalgae has grown tremendously. The immense chemical diversity of microalgae provides numerous applications in the food, feed and pharmaceutical industries. Microalgae are cultivated for the production of whole biomass and valuable substances such as nutraceuticals, carotenoids, phycocyanin and poly-unsaturated fatty acids (PUFAs), which are utilised in the food and feed (notably aquaculture) industry. The production of biofuel from lipid- or carbohydrates-rich microalgae is under way (Ravishankar et al., 2012).

This datasheet deals exclusively with microalgae. For the feed utilization of macroalgae such as kelp, see the Seaweeds datasheet.

Microalgae for feed

Microalgae species appeared in the FAO statistics in 2003 with the Chinese production of several species of spirulina, which was first recorded at 16,483 t and rose sharply to 97,104 t in 2010. These microalgae have gained popularity as a nutraceutical all over the world. They are increasingly used as a dietary ingredient for poultry, and as a protein and vitamin supplement for aquaculture (Muller-Feuga, 2013). Microalgae are required for the nutrition of the larvae of fish and crustaceans, either for direct consumption in the case of molluscs and penaeid shrimp, or indirectly as feed for the live prey fed to small-larvae fish. In all these cases, the post-larvae are hatched, bred, and raised in specialized hatcheries (Muller-Feuga, 2013). As of 1999, aquacultural hatcheries used approximately one-fifth of the 5,000 t of yearly global microalgae production: 62% were molluscs, 21% were shrimp, and 16% were fish (Spolaore et al., 2006). It was estimated that of the 63 million t of aquacultural production in 2005, 35 million t were fed only on pond-grown green water. In 2011, the total ingestion of green water microalgae by cultured fish and crustaceans via planktivory was estimated at 240 million t (Zmora et al., 2013).

In intensive monoculture systems, microalgae are used to feed directly, or indirectly, larval stages of bivalves, shrimp, and some species of fish. Species of algae favoured for this purpose include Chaetoceros, Thalassiosira, Tetraselmis, Isochrysis, Nannochloropsis, Pavlova, and Skeletonema. They are either consumed directly by the target animals (at the larval stage or beyond), or by other organisms such as Artemia, rotifers, and Daphnia, which are, in turn, fed to the target larval organisms. Nutriments such as fatty acids and vitamins are consumed by intermediary zooplankton and transferred to higher trophic levels (the larvae and other stages of fish, shrimp and molluscs) (Becker, 2013b).

In extensive polyculture systems, microalgae are used for the growth of bivalves, carp, and shrimp. This type of aquaculture is becoming advanced in developing countries, and is contributing significantly to the protein supply of the most populous countries, especially India and China. Species with different feeding requirements are reared together. Faeces from carnivorous species support phytoplankton and zooplankton blooms, which in turn are utilized by filter feeders such as carp and shrimp. The production of algal biomass was estimated at 70 million t (DM) in 2011 (Muller-Feuga, 2013).

Over the last decades, several hundred microalgal species have been tested as feed, but only about 40 species are grown in intensive cultivation systems. These species include diatoms, flagellated and chlorococcalean green algae as well as filamentous blue-green algae, notably spirulina (Becker, 2013b).

Table 1. Algal species and their applications in aquaculture (Becker, 2013b)

Spirulina (Arthrospira)

Among all the algae employed in commercial aquaculture, the cyanobacterium spirulina (Arthrospira sp.) has the broadest range of applications. Arthrospira is a microscopic blue-green alga in the shape of a spiral coil, living both in sea and fresh water as a free-floating filamentous organism characterized by cylindrical, multicellular trichomes in an open left-hand helix. Spirulina is the common name for human and animal food supplements produced primarily from two Arthrospira species: Arthrospira platensis, which occurs in Africa, Asia and South America, and Arthrospira maxima, which is confined to Central America. They occur naturally in tropical and subtropical lakes with high pH and high concentrations of carbonate and bicarbonate. Arthrospira is cultivated around the world. Its main applications are human dietary supplements and whole food, and feed supplements in the aquaculture, aquarium, and poultry industries (Becker, 2013b). Spirulina has a long history of being used for food in Chad, as far back as the 9th century Kanem Empire. It is still in daily use today, dried into cakes called dihé, harvested from small lakes and ponds around Lake Chad (Abdulqader et al., 2000; Garofalo, 2011).

Processes

Preservation

As marine invertebrates depend on microalgae for their whole life cycle, mollusc or fish hatcheries need to include a microalgae production system in parallel to their fish production. However, handling live microalgae is not without problems and various attempts have been made to replace living algae by processed algae-based diets with a longer shelf life (Becker, 2013b).

Spray-drying

Spray-drying is probably the most common technique used to preserve microalgae as a sole feed or supplement to the diet in aquaculture (Becker, 2013b). The shelf life is up to 2 years if stored at a low temperature (Zmora et al., 2013). Spray-dried Chlorella vulgaris and Nannochloropsis sp. are available as intact cells or broken cell walls (Zmora et al., 2013).

Freeze-drying

Freeze-drying is a good method for producing an algal feed with a shelf life similar to that obtained with spray-drying, resulting in diets that are almost identical to fresh diets regarding size, shape, and biochemical composition (Becker, 2013b).

Low temperatures

Refrigerated (0-4°C) concentrated pastes of 90-300 g/L (DM) have a shelf life ranging from 4 weeks to 1 year. All algal pastes are nonviable cells except for Chlorella vulgaris (130 g/L). Frozen paste kept at -20°C may have a shelf life of up to 2 years. Cultures of Nannochloropsis oculata have been kept alive at 8°C for over 100 days, or at 5°C for 18 months when provided with light and nitrogen (Zmora et al., 2013).

Microencapsulation

The encapsulation of microalgae in digestible microcapsules allows the delivery to suspension feeders without loss of nutrients to the aqueous medium. The stabilization of the capsule was best when microalgae were cultivated in 2% CaCl2 concentration (Joo et al., 2001).

Cell walls disruption

Most microalgae contain cell walls that are detrimental to digestion in monogastric species. Treatment is necessary to disrupt the cell walls: physical methods include boiling and various types of high temperature drying, if necessary simply sun drying; chemical methods include autolysis or breaking of hydrogen bonds by phenol, formic acid, or urea (which requires later detoxification) (Becker, 2013a).

Live vs. dried microalgae

Dried microalgae are often less efficient than live microalgae for feeding marine larvae. The oxidation of microalgae that occurs during drying causes a loss of highly unsaturated fatty acids essential for larval growth. Dried cells tend to disintegrate when kept in suspension. Due to the broken cell walls, water-solubles components are lost in the culture media and are no longer available to the host species. Dried microalgae are also more susceptible to pathogenic bacterial contamination. Algae paste exhibit similar issues. These problems can be alleviated by proper preparation (centrifugation, flocculation, or filtration) and/or preservation techniques (additives and freezing) that preserve cell wall integrity (Becker, 2013b).

Forage management

Open cultivation systems

Most algae for commercial use are grown in the open air. The two most common open cultivation systems are circular and raceway ponds. These systems can be developed using natural water bodies such as lagoons and ponds, or artificial ponds such as raceways. Open systems, such as coastal shallow brackish-water ponds, are extensively used for feed production in aquaculture and for other industrial applications. The open cultivation pond is cheap and they do not compete for agricultural land as they can be built on non-productive (marginal) land, or coastal regions for marine algal cultivation. Open systems require minimal investment in terms of light source and operations. However, open systems provide less control over abiotic conditions (rainfall, temperature, dust, etc.) and have a greater risk of contamination by other algae species, or by microorganisms such as rotifers (Ravishankar et al., 2012). In some countries, notably in Asia, microalgae are used in integrated livestock management in manure ponds for growing fish, thereby providing feed and avoiding use of fish meal (Phang, 1992).

Closed cultivation systems

Commercial algae production for aquaculture, particularly for hatcheries, often relies on closed cultivation systems where culture parameters can be closely controlled. Such systems have a high biomass productivity compared with open ponds since culture parameters such as light, turbulence and air exchange can be carefully regulated (Ravishankar et al., 2012). The two main closed systems are:

Photobioreactors, which provide environments with the greatest level of control. They include tubular systems, flat-plate vessels, vertical cylinders and vertical or horizontal bag cultures.

Fermenters, which are used for large-scale production of freshwater (Chlorella vulgaris, in South-East Asia) and marine algae (Schizochytrium sp. and Crypthecodinium cohnii) (Zmora et al., 2013).

Green water technique

Another technique, called the green water technique, consists in introducing and maintaining some algae cultures in larval-rearing tanks. It is a common practice in hatcheries of most cultured species, including fish, shrimp, and crabs, that do not consume algae directly (Zmora et al., 2013). The addition of microalgae to rearing ponds helps to stabilize and improve the quality of the medium. It often results in better survival and growth rates than the clear-water technique, probably due to oxygen production and pH stabilization. In addition, algal compounds regulate and control bacterial contamination (Becker, 2013b).

Environmental impact

Water systems containing organic materials, such as swine waste, fishery waste, etc. can be used to grow microalgae. Using algae in this way has been proposed as a means of reducing biological oxidation demand in water systems and improving water quality, while at the same time generating a supplemental protein source that can be used in feeding livestock (Goh, 1989).

Nutritional aspects

Nutritional attributes

Microalgae vary significantly in the composition of their constituents. These differences may reflect genetic differences as well as culture conditions and the growth stage at harvest (Brown et al., 1997; Becker, 2013b). In the case of marine or freshwater species, it has been noted that the growth of those fed with mixtures of several algal species is often superior to that obtained by feeding only one species, probably because a particular alga may lack a nutrient that another may contain (Yamaguchi, 1996). Thus, it has been recommended to feed animals with mixtures of carefully selected microalgae for optimal growth (Becker, 2013b).

Protein

Microalgae are usually rich in protein, which may amount to more than 60% of DM. Spirulina (Arthospira) contains a high amount of protein, between 55 and 77% of DM (Garofalo, 2011). As noted above, there are considerable variations due to genetic or culture conditions. For instance, some Chlorella strains contain less than 30% protein in the DM, whereas regular Chlorella exceed 50% protein. The protein content was found to be more susceptible to medium-induced variation than the other cellular constituents (Becker, 2013b).

Amino acids

The amino acid composition of microalgal proteins is quite similar between species, and relatively unaffected by intrinsic and extrinsic factors (Becker, 2013b). For example, cultures grown over a range of light intensities were identical in amino acid composition, as were those grown in stationary phase and logarithmic-phase cultures (Brown et al., 1993). In general, aspartic acid and glutamic acid occur in the highest concentrations, and cysteine, methionine, tryptophan, and histidine occur in the lowest concentrations (Becker, 2013b). Spirulina protein contains all essential (for humans) amino acids, though reduced amounts of methionine, cysteine, and lysine when compared to animal proteins. It is superior to a typical plant protein, including that of legumes (Garofalo, 2011).

Lipids

The quality of the algal lipids is of prime importance to the nutritional value of microalgae in aquaculture. Fatty acids from microalgae may be efficiently transferred to higher trophic levels (e.g. to fish larvae) via zooplankton. Polyunsaturated fatty acids (PUFAs), in particular eicosapentaenoic acid (EPA, C20:5 n-3), arachidonic acid (AA, C20:4 n-6), and docosahexaenoic acid (DHA, C22:6 n-3) are of major importance in the evaluation of the nutritional composition of an algal species. C18:2 n-6 and/or C18:3 n-3 (α-linolenic acid, ALA) are essential for many freshwater fish. C20 PUFAs can form longer chains more efficiently in freshwater fish than in marine fish (Becker, 2013b). Generally, the fatty acid content in microalgae shows systematic differences according to the taxonomic group, although there are always differences between species from the same algal class (Becker, 2013b).

Most microalgal species have moderate to high percentages of EPA. Diatoms, eustigmatophytes, cryptomonads, rhodophytes, and some prymnesiophytes (Pavlova spp.) are all rich sources of EPA (7-34%) (Volkman et al., 1993). Many representatives from these classes have been used successfully as feed in larval culture (Brown et al., 1989).

Prymnesiophytes (e.g., Pavlova spp. and Isochrysis sp.) and cryptomonads are relatively rich in DHA, whereas eustigmatophytes (Nannochloropsis spp.) and diatoms have the highest percentages of AA.

Chlorophytes (Dunaliella spp. and Chlorella spp.) are deficient in both C20 and C22 PUFAs, although some species have small amounts of EPA. Because of this PUFA deficiency, chlorophytes generally have low nutritional value and are not suitable when used alone in the diet (Brown et al., 1989).

Cryptomonads and prymnesiophytes are relatively rich in DHA, whereas eustigmatophytes, rhodophytes, and diatoms are highest in AA.

Prasinophyte species contain significant proportions of C20 and C22 PUFAs.

Prasinophyte species such as Tetraselmis spp. have been used successfully for prawn and mollusc culture (Brown et al., 1992b).

Significant levels of C18:2 n-6 and C18:3 n-3 are found in most microalgal groups, except diatoms and eustigmatophytes which contain very low levels (Volkman et al., 1993).

Large variations do occur in fatty acid composition. For instance, in spirulina of different origins, palmitic acid, C16:0, ranged from 18 to 39%; oleic acid, C18:1, from 3 to 20%; linoleic acid, C18:2 n-6, from 6 to 16%; and γ-linolenic acid from 4 to 23% (Diraman et al., 2009).

Different strategies are applied to improve the PUFA content in microalgae. Manipulation of processing conditions such as light intensity, nutrient status, or temperature allows the modulation of the lipid composition and consequent optimization of their overall yield and productivity (Becker, 2013b).

Fibre

With the exception of the cyanobacteria spirulina (Arthrospira) and Aphanizomenon flos-aquae, most microalgae possess a relatively thick cellulosic cell wall, which poses a problem in digesting algal biomass by monogastric species. Treatments are necessary to disrupt the cell wall, and make the algal protein nutritionally accessible (see Processes in the "Description" tab). The cell wall of spirulina does not represent a barrier to proteolytic enzymes, and this alga can be digested by monogastrics without previous physical or chemical rupture of the cell wall (Becker, 2013a).

Vitamins

Algae generally provide excess or adequate amounts of vitamins to support normal growth in aquacultural species (Brown et al., 1999). However, a comparison of the vitamin content of 5 microalgae species (Tetraselmis suecica, Isochrysis galbana, Pavlova lutheri, Skeletonema costatum and Chaetoceros calcitran) showed that though all five were rich in most vitamins, they also had low concentrations of at least one or more (De Roeck-Holtzhauer et al., 1991). Therefore, mixed algal diets may be necessary to meet the vitamin requirements of maricultured species or zooplankton. Transfer of vitamins between trophic levels is important for fish larvae and late larval/early juvenile crustaceans that are reared on algal-fed zooplankton (Becker, 2013b).

The levels of ascorbic acid (vitamin C) in 11 microalgal species during logarithmic and stationary growth phases were found to range 15-fold and to be unrelated to algal class. Values ranged from 1.1 g/kg DM (Thalassiosira pseudonana) to 16 g/kg (Chaetoceros muelleri). Many of the species had different levels of ascorbic acid between logarithmic and stationary phases (Brown et al., 1992a). These values were above the requirement of mariculture species (Becker, 2013b). A similar study on the riboflavin content of 6 species found that the concentrations at the logarithmic phase ranged from 20 mg/kg DM (Thalassiosira pseudonana) to 40 mg/kg (Isochrysis sp.). Riboflavin increased in all species in the stationary growth phase, sometimes double or threefold. Chaetoceros gracilis contained more riboflavin (106 mg/kg) than all other species (48-61 mg/kg) in the stationary phase (Brown et al., 1994). Levels in all species were in excess of the dietary requirements of maricultured species (Becker, 2013b).

Pigments

Microalgae are an important source of pigments, notably carotenoids such as ß-carotene, lutein and astaxanthin. Spirulina (Arthrospira) contains many pigments, including chlorophyll a, ß-carotene, echinenone, myxoxanthophyll, zeaxanthin, canthaxanthin, diatoxanthin, 3'-hydroxyechinenone, ß-cryptoxanthin, oscillaxanthin, plus the phycobiliproteins C-phycocyanin and allophycocyanin (Leema et al., 2010). A survey of the carotenoid content of cultures of 15 Chlorophycean microalgae found that lutein was the most abundant carotenoid in all strains except one. The highest lutein levels were found in Chlorella fusca SAG 211-8b, Chlorococcum citriforme, Neospongiococcum gelatinosum, and Muriellopsis sp. Violaxanthin and ß-carotene were found in virtually all the strains tested, although at lower levels than those of lutein. The most important factors that affect lutein content in microalgae are temperature, irradiance, pH, availability and source of nitrogen, salinity (or ionic strength), and the presence of oxidizing substances (or redox potential) (Becker, 2013b).

The green microalga Haematococcus pluvialis represents the richest biological source of astaxanthin, and is the only source for microalgal astaxanthin. Haematococcus hematocysts contain 1.5-3% of the dry biomass after the reddening phase. After harvesting, the biomass is dried and cracked to fracture the thick, hard cell wall of the cysts, thus ensuring maximum bioavailability (Becker, 2013b). Astaxanthin is used as a pigmentation source in aquaculture, as a vitamin A precursor in fish, as well as an enhancer of the immune system of fish and shrimp, for maximum survival and growth. Natural micro-algal astaxanthin has shown superior bio-efficacy over the synthetic form (Ravishankar et al., 2012).

Feeding experiments with microalgae and ruminants are scarce, since large amounts of algal biomass are required and the technical requirements are high. While some results are positive, the effect of feeding microalgae to ruminants remains inconclusive (Becker, 2013a).

Dairy cows

In Lithuania, dairy cows fed a forage diet supplemented with 2 g/d of fresh spirulina, Arthrospira platensis, for 2 months exhibited higher milk yield (+7.6%), higher percentages of milk fat (+18-25%), milk protein (+10%) and lactose (+12%), and a large decrease in the number of somatic cells (-29%) (Simkus et al., 2007). However, it has been pointed out that such a small amount of wet algae is unlikely to cause such dramatic responses (Becker, 2013a). In another experiment in Lithuania, dairy cows fed 200 g/d of dry spirulina, Arthrospira platensis, for 3 months became 8.5-11% fatter, and produced 6 kg/d more milk than those in the control group (Kulpys et al., 2009). In Greece, dairy cows fed 40 g/d of powdered Arthrospira platensis for 7 weeks produced milk with decreased saturated fatty acids, and increased monounsaturated and polyunsaturated fatty acids (Christaki et al., 2012). In Canada, dairy cows fed maize silage supplemented with 1.1 to 4.2 kg/d pellets containing 55% of defatted algae, Crypthecodinium cohnii, showed increased trans-18:1 isomers in rumen fluid, probably due to the inhibition of the reduction of trans-18:1 to 18:0 by algae extract. The trans-18:1 isomers serve as precursors for CLA biosynthesis in the tissues of ruminants (Or-Rashid et al., 2008).

Beef cattle

In the USA, dried sewage-grown algae Chlorella spp., Scenedesmus obliquus or Scenedesmus quadricauda, fed to steers in pelleted rations composed of algae and alfalfa hay (at 20:80 and 40:60) resulted in similar DM and OM digestibilities as diets based on alfalfa hay only (Hintz et al., 1966). In Bangladesh, a liquid suspension of Chlorella spp. and Scenedesmus spp. given to growing indigenous cattle was drunk at 10% of live weight, and compared to a supplement of 0.5 kg/d sesame oil cake. The inclusion of algal suspension did not improve metabolizable energy, protein intake and OM digestibility, but slightly increased daily gain and significantly improved fibre digestibility (Chowdhury et al., 1995). In Bulgaria, for 8-month old bulls given the same feed without or with 1 L centrifuged Scenedesmus acutus in suspension, the algal treatment did not affect digestibility but it increased the relative proportion of digestion occurring in the intestines, and increased blood red cells and carotene content (Ganovski et al., 1975). In Iran, Holstein calves fed 2, 6, or 25 g/d of dried spirulina Arthrospira platensis for 2 months did not show significant differences in final weight, daily gain, daily feed intake and feed efficiency, but the highest level decreased nutrient digestibility and plasma cholesterol concentration (Heidarpour et al., 2011).

Sheep

Wethers and lambs

In the USA, air-dried or drum-dried sewage-grown algae Chlorella spp., Scenedesmus obliquus or Scenedesmus quadricauda, fed to wethers in diets composed of algae/alfalfa hay (60:40) resulted in protein digestibility of about 70% for all diets including the control. However, digestibility of carbohydrate was lower for the algae-based diets, which may be explained by the lower digestibility, even for ruminants, of algal cell walls. Algae were poorly palatable: when the diet was unpelleted, the sheep sorted the feed and left the algae. For weaning lambs fed cottonseed meal, alfalfa or alfalfa/algae pellets, alfalfa pellets were unsatisfactory, whereas with cottonseed meal and alfalfa/algae pellets the animals were able to maintain or gain weight (Hintz et al., 1966). In Australia, a mixture of sewage-grown algae, filtration paper and barley (algae+paper:barley 30:70) was compared with alfalfa in sheep: the algae/paper mixture was inferior to the alfalfa (Davis et al., 1975). In Brazil, sheep fed spirulina Arthrospira platensis up to 1.8% (DM basis) of the diet did not show differences in performance, nutrient intake, nutrient digestibility, carcass traits, proportions of leg tissues and their muscularity (Suassuna, 2014).

Ewes

In the UK, ewes supplemented with spray-dried, DHA-rich algae Crypthecodinium cohnii at 64 g/d gave birth to more vigorous lambs compared to unsupplemented ewes. The amount of vigour depended on the duration of supplementation before lambing. After parturition concentrations of DHA and eicosapentaenoic acid (EHA) in ewe and lamb plasma and colostrums were proportional to the length of the supplementation period (Pickard et al., 2008).

Pigs

There have been few experiments with pigs. Microalgal biomass appears to be palatable for pigs, of acceptable nutritional quality and able to partly replace soybean meal or fish meal (Becker, 2013a).

In Japan, the addition of 2% freeze-dried Chlorella powder to the diet of young pigs had a favourable effect on growth and feed efficiency (Tamiya, 1961). In the USA, air-dried or drum-dried sewage-grown algae Chlorella spp., Scenedesmus obliquus or Scenedesmus quadricauda, fed to growing pigs at 2.5, 5, or 10% of the diet, substituted for soybean meal, resulted in a similar performance to that of the control diet. Algae also supplied adequate protein to supplement barley for growing-finishing pigs (Hintz et al., 1966). In another series of trials, the same algae meal supplemented with some B vitamin sources adequately replaced fish meal, in isonitrogenous diets based on barley and fish meal, without depressing daily gain and/or feed efficiency. When vitamin B12 was omitted from the algal diet, there was a decrease in daily gain. Carcass characteristics were identical between pigs fed the algal diets and those fed diets containing fish meal. The digestible energy of algae was low but algal protein was 70% digestible (Hintz et al., 1967). Algae meal from Spirulina maxima, Arthrospira platensis and Chlorella spp. replaced up to 50% of soybean protein (and 33% of dietary protein) in the diets of early weaned pigs without depressing performance or causing health issues (diarrhoea, loss of appetite, toxicity, histopathological lesions). Pigs fed with the algal diet showed an improvement in weight gain, and pigs fed with 1 g/kg algae tended to have the best feed efficiency (Yap et al., 1982).

Poultry

Microalgae can be exploited as poultry feed, both for their protein and their carotenoid contents, which are necessary to enhance pigmentation in poultry meat and eggs. Microalgal biomass up to a level of 5-10% of the diet can be used safely as a partial replacement of a conventional protein source, but higher concentrations may cause adverse effects if the feeding period is prolonged (Spolaore et al., 2006).

Broilers and growing chickens

Inclusion of up to 6% sewage-grown, alum-flocculated Micractinium algae meal in well-balanced broiler diets from 1 to 7 weeks of age had no adverse effect either on growth or the feed/gain ratio. However, a reduced feed intake in young chicks fed 9% algae resulted in a graded decrease in weight gain, while in the finisher period the lower feed intake caused a reduction in the accumulation of abdominal fat (Lipstein et al., 1981). Sewage-grown Chlorella or Micractinium algae included at 5 and 10% in balanced diets were found to be suitable protein supplements in broiler diets, and had no adverse effect on growth, feed efficiency, or carcass fat (Lipstein et al., 1983). In India, supplementation of broiler diets with sun-dried spirulina Arthrospira platensis replacing either fish meal or groundnut cake at an isonitrogenous concentration of 14% and 17% did not alter feed efficiency, dressing percentage growth performance, or histopathology. Meat quality remained unchanged except for a more intensive colour in the case of birds fed on the algal diet (Venkataraman et al., 1994). Two experiments in Hawaii have been less favourable. In the first one, cockerel chicks fed isonitrogenous diets containing 10 or 20% dried spirulina Arthrospira platensis showed depressed growth at 3 weeks of age, although feed efficiency was not affected. In the second experiment, broiler chicks fed 12% of spirulina grew slower than the birds fed the control diet or up to 6% spirulina (Ross et al., 1990). A Chlorella strain with very thin cell walls was found to be low in protein (less than 25% of DM) but very palatable and digestible in chicks, and could be used safely at inclusion rates as high as 20% (Yoshida et al., 1982).

In Germany, organic chickens supplemented with 1.25 to 5% dried spirulina Arthrospira platensis consumed the mixtures without problems, did not exhibit health issues, and had higher carcass weights and yield. However, the high cost of algae meal made their use economically ineffective (Bellof et al., 2010).

Spirulina Arthrospira platensis supplementation in chicks at 1% of the diet increased several immunological functions, implying that a dietary inclusion of spirulina may enhance disease resistance potential in chickens (Qureshi et al., 1996).

Laying hens

Microalgae have been used in layer diets to improve yolk colour. Chlorella vulgaris is rich in lutein and can be used as a feed supplement to improve yolk properties. Diets containing up to 12% of Chlorella sp. algae meal with supplementary DL-methionine did not affect egg output, feed conversion or egg-shell quality. High concentrations of algae meal caused a deep yellow yolk colour of acceptable appearance. Thus algae meal at the concentrations tested can be a useful substitute for soybean meal in diets for laying hens (Lipstein et al., 1980). Another micro-alga widely used for enriching yolk colour is Haematococcus pluvialis, which is rich in astaxanthin. Astaxanthin has been shown to reduce chick mortality by 50%, and to reduce Vibrio spp. infections in eggs, thereby improving their nutritional value (Ravishankar et al., 2006).

Quails

Japanese quails fed up to 12% of dried spirulina Arthrospira platensis did not show altered growth, egg production, egg quality, and hatchability. Yolk colour increased with each succeeding level of spirulina, and fertility was higher for all spirulina treatments compared to the control (Ross et al., 1990).

Rabbits

There are few reports of feeding trials with rabbits. In Italy, growing rabbits fed diets containing up to 15% dried spirulina Arthrospira platensis as a substitute for soybean meal and alfalfa did not show differences in growth rate, feed efficiency, carcass yield, in the proportions of carcass components, and in the chemical composition of the meat, with the exception of the lipid content, which was higher for the spirulina diets. The spirulina inclusion level of 10% gave the highest feed intake. The content of γ-linolenic acid increased in the perirenal fat and meat as the level of algal supplementation increased. Spirulina could thus potentially be used in rabbit nutrition with benefits on the nutritional quality of rabbit meat for consumers (Peiretti et al., 2008; Peiretti et al., 2011). The inclusion of 12% of green alga Scenedesmus acutus (54% crude protein and 12% lipid) as a protein replacement for soybean meal decreased total intake and weight gain. The reduced rate of gain was proportional to a drop in protein digestibility, and consequently to a lower availability of amino acids (Battaglini et al., 1979; Grandi et al., 1986).

Fish

Microalgae play a vital role in the rearing of both carnivorous and herbivorous fish. They are used to supply basic nutrients, and as a source of pigments to colour the flesh of salmonids or the skin of ornamental fish, or for other biological purposes (Becker, 2013b). The good nutrient profiles of algae, and their contents in carotenoids and PUFAs, can improve fish quality considerably (Phang, 1992). In developing countries, microalgae such as spirulina can be grown on a small scale in rural areas and may help to improve local aquaculture, for instance by replacing expensive protein sources such as fish meal (Becker, 2013b).

Marine small-larvae fish require small, live, plankton feeder preys, namely the rotifers, Brachionus plicatilis and Brachionus rotundiformis, which require microalgae. Microalgae allow a quick recovery of rotifer populations after a collapse in numbers, improve the nutritional quality of live prey, and lower bacterial contamination (Muller-Feuga, 2013). For marine fish larvae, the most popular microalgae for boosting zooplankton intermediates with PUFAs are those that contain high levels of 20:5 (n-3) (e.g. Nannochloropsis oculata), 22:6 (n-3) (e.g. Isochrysis sp.), or both (e.g. Pavlova lutheri). Rotifers (Brachionus plicatilis) readily ingest Pavlova cells, from which they can accumulate high concentrations of 20:5 (n-31), 22:6 (n-3), and other PUFAs (Becker, 2013b). Some freshwater cyprinids and cichlids are herbivorous and some are plankton feeders. They are reared in well-integrated polyculture systems where cyprinids of different feeding requirements, such as silver carp (Hypophthalmichthys molitrix) or bighead carp (Hypophthalmichthys nobilis), are stocked together at ratios such that feed resources are fully utilized (Muller-Feuga, 2013). Spirulina Arthrospira platensis has been extensively used in rearing some fish species, including red seabream (Pagrus major), cherry salmon (Oncorhynchus masou), nibbler (Girella punctata), striped jack (Pseudocaranx dentex), yellowtail (Seriola quinqueradiata) and Mozambique tilapia (Oreochromis mossambicus), to improve weight gain, muscle protein deposition, raw meat quality, flesh texture and taste (Hasan et al., 2009).

Salmonids

The predominant source of carotenoids for salmonids has been synthetic carotenoids like astaxanthin, which has been used for pigmentation since the 1990s (Ravishankar et al., 2006). Natural sources of astaxanthin for commercially raised salmonids include processed crustacean waste from krill, shrimp, crab and crawfish. However, crustacean waste products contain large amounts of moisture, ash and chitin, which limit their use in salmon feed. The efficiency of dietary astaxanthin using microalgae for flesh pigmentation of Atlantic salmon and rainbow trout has been demonstrated (Torrissen et al., 1989). Astaxanthin is also considered as a vitamin for salmon, as it is essential for the proper development and survival of juveniles. Dried Haematococcus pluvialis fed to rainbow trout up to 6% of the diet had no major effect on growth or mortality and was found a safe and effective source of pigment (Choubert et al., 1993).

Carp

The potential of spirulina as feed for carp fry was tested in central India on six carp species including silver carp (Hypophthalmichthys molitrix), grass carp (Ctenopharyngodon idella), and the common carp (Cyprinus carpio). The fish were fed a control diet composed of a mixture of local feed ingredients such as groundnut cake and rice bran or with the addition of 10% spirulina. Grass carp and common carp were also studied when feeding them live algae. In almost all tests, addition of spirulina resulted in better performance (Ayyappan, 1992). Supplementation of Spirogyra spp. for carp (Catla catla) improved growth, muscle protein and fat content (Kumar et al., 2004). Likewise, common carp fed diets in which different amounts of fish meal protein were replaced by Arthrospira platensis, up to total replacement showed no negative effects on final weight, specific growth rate, feed conversion ratio, protein efficiency ratio, or organoleptic evaluations (Nandeesha et al., 1998).

Microalgae supplementation has been found useful for pigmentation. The skin colour of ornamental koi carp fish increased considerably when fed a diet containing astaxanthin enriched Haematococcus pluvialis cells at 25 mg/kg in the feed (Kamath, 2007). It was also useful for the reduction of metal toxicity. In Labeo rohita carp exposed to toxic concentrations of copper (up to 0.5 ppm), the addition of up to 10% spirulina improved the physiological and biochemical parameters and reduced the metal burden in carp tissues. Levels above 4% did not result in better performance or higher phosphatase activity. However, the treatment period (21 days) was not sufficient for complete removal of the copper (James, 2010).

Tilapia

In larval Nile tilapia (Oreochromis niloticus) fed different amounts of fresh spirulina Arthrospira platensis cultivated in photobioreactors, acceptability tended to improve with an increase in the initial length of the larvae, and 2 cm larvae fed spirulina showed significantly rapid growth. Best growth was obtained when there was an abundant supply of spirulina during the early stages (Lu et al., 2002). Tilapia fed spirulina during their larval stage maintained normal reproduction from parents to progeny through three generations (Lu et al., 2004). Mozambique tilapia (Oreochromis mossambicus) fry were fed for 9 weeks a diet where fish meal was replaced partly or fully with spirulina Arthrospira maxima. The growth rate and protein utilization of fish fed the diet with 20% or 40% spirulina protein (about 10 or 20% of the diet) were not significantly different from those fed the control diet. Further increases in the algae protein content decreased growth and feeding performance (Olvera-Novoa et al., 1998). A similar result was obtained with hybrid red tilapia (Oreochromis mossambicus × Oreochromis niloticus) where spirulina Arthrospira platensis included at 20% dietary level in partial substitution for fish meal did not affect final weight gain, specific growth rate, feed conversion ratio, and survival rate of fish (Ungsethaphand et al., 2010).

The use of spirulina Arthrospira platensis as a growth and immunity promoter was evaluated in an experiment where Nile tilapia fry were fed diets containing up to 1% spirulina and then challenged with the pathogenic bacterium Aeromonas hydrophila. Optimum growth and feed utilization were obtained with 0.5% spirulina. Total fish mortality due to infection with Aeromonas hydrophila decreased with an increase in the spirulina level in the diet. Fish fed on diets containing 0.5-1% spirulina exhibited higher red and white blood cells counts, glucose, lipids, protein, albumin and globulin, indicating that spirulina supplementation could be an alternative method to using antibiotics for disease prevention (Abdel-Tawwab et al., 2009).

Microalgae are necessary for the second stage of larval development (zoea) and in combination with zooplankton for the third stage (myses) of the development of shrimps. Although of short duration, those larval stages require microalgae culture facilities, which will vary with the size of the hatchery and the level of growth control. The larvae feed consists of a combination of microalgae and early stages of the phyllopod crustacean Artemia sp., as well as dry food available on the market, occasionally manufactured locally (Muller-Feuga, 2013). The most common species used to feed shrimp larvae are Chaetoceros calcitrans, Chaetoceros muelleri, Chaetoceros gracilis and Skeletonema sp. The species used for crab larvae are Nannochloropsis sp., freshwater Chlorella vulgaris B12 (a strain that absorbs the B12 vitamin) and marine Chlorella (Zmora et al., 2013).

Other species

Molluscs

Mollusc production is of all aquatic productions the highest consumer of microalgae. Filtering molluscs such as oysters, clams, mussels, and pectinids are herbivorous and consume microalgae from post-larval, and sometimes larval, stages. They are suspension feeders, taking in plankton composed of plant or animal particles. This production generally relies on wild phytoplankton present in the water masses. When larvae and post-larvae are produced in hatcheries, fodder microalgae produced artificially must be added to meet the feed requirements of larvae, post larvae, and broodstock (Muller-Feuga, 2013).

Algae rich in PUFAs and carbohydrates are reported to produce the best growth for juvenile oysters and larval scallops (Patinopecten yessoensis) (Whyte et al., 1989). High dietary protein provides the best growth for juvenile mussels (Becker, 2013b). Mollusc larvae tend to grow better with live microalgae, whose natural bacterial flora is beneficial to mollusc health. Replacement with preserved algae paste, non-living algae, microencapsulated diets, or spray-dried algae can only be partial (Becker, 2013b). In juvenile clams (Tapes semidecussata) and Pacific oysters (Crassostrea gigas), up to 40% of a live algal diet (Tetraselmis suecica and Chaetoceros sp.) was replaced with dried Schizochytrium without a significant reduction in growth rate (Boeing, 1997). However, full replacement was possible with juvenile mussels Mytilus galloprovincialis, who were cultured on a spray-dried algal diet of 50% Schizochytrium sp. and 50% Arthrospira platensis without reducing growth. Growth of mussels fed spray-dried diets composed of mixtures of Schizochytrium sp., spirulina or Haematococcus pluvialis was not significantly different from that of mussels fed an equal ration (DM) of a mixed diet of living Isochrysis galbana and Chaetoceros calcitrans (Langdon et al., 1999). Abalone (Haliotis discus) showed a significantly higher growth when fed diets based on fish meal and spirulina than those fed diets prepared with soybean meal and tortula yeast (Stott et al., 2004). Cooled microalgal paste composed of Skeletonema costatum and Chaetoceros calcitrans used as supplement for the Pacific oyster was as effective as live algae (McCausland et al., 1999).

In France, oysters are put in contact with naturally or artificially grown diatom Haslea ostrearia, so that they acquire a blue-green colour on the gills and labial palps. This technique called "greening" (verdissage) raises the product’s market value by about 30% (Muller-Feuga, 2013).